scholarly journals Differential Effects of β1 and β2 Subunits on BK Channel Activity

2005 ◽  
Vol 125 (4) ◽  
pp. 395-411 ◽  
Author(s):  
Patricio Orio ◽  
Ramon Latorre
Keyword(s):  
Steady State ◽  
Calcium Binding ◽  
Voltage Dependence ◽  
Calcium Sensitivity ◽  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensitivity ◽  
Channel Activation ◽  
Α Subunit ◽  
Voltage Sensors

High conductance, calcium- and voltage-activated potassium (BK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory (β) subunits. β1 and β2 subunits increase apparent channel calcium sensitivity. The β1 subunit also decreases the voltage sensitivity of the channel and the β2 subunit produces an N-type inactivation of BK currents. We further characterized the effects of the β1 and β2 subunits on the calcium and voltage sensitivity of the channel, analyzing the data in the context of an allosteric model for BK channel activation by calcium and voltage (Horrigan and Aldrich, 2002). In this study, we used a β2 subunit without its N-type inactivation domain (β2IR). The results indicate that the β2IR subunit, like the β1 subunit, has a small effect on the calcium binding affinity of the channel. Unlike the β1 subunit, the β2IR subunit also has no effect on the voltage sensitivity of the channel. The limiting voltage dependence for steady-state channel activation, unrelated to voltage sensor movements, is unaffected by any of the studied β subunits. The same is observed for the limiting voltage dependence of the deactivation time constant. Thus, the β1 subunit must affect the voltage sensitivity by altering the function of the voltage sensors of the channel. Both β subunits reduce the intrinsic equilibrium constant for channel opening (L0). In the allosteric activation model, the reduction of the voltage dependence for the activation of the voltage sensors accounts for most of the macroscopic steady-state effects of the β1 subunit, including the increase of the apparent calcium sensitivity of the BK channel. All allosteric coupling factors need to be increased in order to explain the observed effects when the α subunit is coexpressed with the β2IR subunit.

scholarly journals Structural Determinants for Functional Coupling Between the β and α Subunits in the Ca2+-activated K+ (BK) Channel

2006 ◽  
Vol 127 (2) ◽  
pp. 191-204 ◽  
Author(s):  
Patricio Orio ◽  
Yolima Torres ◽  
Patricio Rojas ◽  
Ingrid Carvacho ◽  
Maria L. Garcia ◽  
...  
Keyword(s):  
Calcium Sensitivity ◽  
Bk Channels ◽  
Bk Channel ◽  
Fine Tuning ◽  
Functional Coupling ◽  
Xenopus Laevis Oocytes ◽  
Voltage Sensitivity ◽  
Patch Clamp Technique ◽  
Α Subunit ◽  
Intracellular Domains

High conductance, calcium- and voltage-activated potassium (BK, MaxiK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory (β) subunits. The most remarkable effects of β1 and β2 subunits are an increase of the calcium sensitivity and the slow down of channel kinetics. However, the detailed characteristics of channels formed by α and β1 or β2 are dissimilar, the most remarkable difference being a reduction of the voltage sensitivity in the presence of β1 but not β2. Here we reveal the molecular regions in these β subunits that determine their differential functional coupling with the pore-forming α-subunit. We made chimeric constructs between β1 and β2 subunits, and BK channels formed by α and chimeric β subunits were expressed in Xenopus laevis oocytes. The electrophysiological characteristics of the resulting channels were determined using the patch clamp technique. Chimeric exchange of the different regions of the β1 and β2 subunits demonstrates that the NH3 and COOH termini are the most relevant regions in defining the behavior of either subunit. This strongly suggests that the intracellular domains are crucial for the fine tuning of the effects of these β subunits. Moreover, the intracellular domains of β1 are responsible for the reduction of the BK channel voltage dependence. This agrees with previous studies that suggested the intracellular regions of the α-subunit to be the target of the modulation by the β1-subunit.

scholarly journals Molecular mechanism of BK channel activation by the smooth muscle relaxant NS11021

2020 ◽  
Vol 152 (6) ◽  
Author(s):  
Michael E. Rockman ◽  
Alexandre G. Vouga ◽  
Brad S. Rothberg
Keyword(s):  
Smooth Muscle ◽  
Muscle Relaxant ◽  
Time Course ◽  
Bk Channels ◽  
Bk Channel ◽  
Neurological Deficits ◽  
Open Probability ◽  
Channel Activation ◽  
Voltage Sensors ◽  
Smooth Muscle Relaxant

Large-conductance Ca2+-activated K+ channels (BK channels) are activated by cytosolic calcium and depolarized membrane potential under physiological conditions. Thus, these channels control electrical excitability in neurons and smooth muscle by gating K+ efflux and hyperpolarizing the membrane in response to Ca2+ signaling. Altered BK channel function has been linked to epilepsy, dyskinesia, and other neurological deficits in humans, making these channels a key target for drug therapies. To gain insight into mechanisms underlying pharmacological modulation of BK channel gating, here we studied mechanisms underlying activation of BK channels by the biarylthiourea derivative, NS11021, which acts as a smooth muscle relaxant. We observe that increasing NS11021 shifts the half-maximal activation voltage for BK channels toward more hyperpolarized voltages, in both the presence and nominal absence of Ca2+, suggesting that NS11021 facilitates BK channel activation primarily by a mechanism that is distinct from Ca2+ activation. 30 µM NS11021 slows the time course of BK channel deactivation at −200 mV by ∼10-fold compared with 0 µM NS11021, while having little effect on the time course of activation. This action is most pronounced at negative voltages, at which the BK channel voltage sensors are at rest. Single-channel kinetic analysis further shows that 30 µM NS11021 increases open probability by 62-fold and increases mean open time from 0.15 to 0.52 ms in the nominal absence of Ca2+ at voltages less than −60 mV, conditions in which BK voltage sensors are largely in the resting state. We could therefore account for the major activating effects of NS11021 by a scheme in which the drug primarily shifts the pore-gate equilibrium toward the open state.

scholarly journals Spontaneous Transient Outward Currents Arise from Microdomains Where BK Channels Are Exposed to a Mean Ca2+ Concentration on the Order of 10 μM during a Ca2+ Spark

2002 ◽  
Vol 120 (1) ◽  
pp. 15-27 ◽  
Author(s):  
Ronghua ZhuGe ◽  
Kevin E. Fogarty ◽  
Richard A. Tuft ◽  
John V. Walsh
Keyword(s):  
Voltage Dependence ◽  
Ryanodine Receptors ◽  
Bk Channels ◽  
Bk Channel ◽  
Channel Activation ◽  
Membrane Area ◽  
Outward Currents ◽  
Cell Recording ◽  
Excised Patches ◽  
Spontaneous Transient Outward Currents

Ca2+ sparks are small, localized cytosolic Ca2+ transients due to Ca2+ release from sarcoplasmic reticulum through ryanodine receptors. In smooth muscle, Ca2+ sparks activate large conductance Ca2+-activated K+ channels (BK channels) in the spark microdomain, thus generating spontaneous transient outward currents (STOCs). The purpose of the present study is to determine experimentally the level of Ca2+ to which the BK channels are exposed during a spark. Using tight seal, whole-cell recording, we have analyzed the voltage-dependence of the STOC conductance (g(STOC)), and compared it to the voltage-dependence of BK channel activation in excised patches in the presence of different [Ca2+]s. The Ca2+ sparks did not change in amplitude over the range of potentials of interest. In contrast, the magnitude of g(STOC) remained roughly constant from 20 to −40 mV and then declined steeply at more negative potentials. From this and the voltage dependence of BK channel activation, we conclude that the BK channels underlying STOCs are exposed to a mean [Ca2+] on the order of 10 μM during a Ca2+ spark. The membrane area over which a concentration ≥10 μM is reached has an estimated radius of 150–300 nm, corresponding to an area which is a fraction of one square micron. Moreover, given the constraints imposed by the estimated channel density and the Ca2+ current during a spark, the BK channels do not appear to be uniformly distributed over the membrane but instead are found at higher density at the spark site.

scholarly journals The Membrane Cholesterol Modulates the Interaction Between 17-βEstradiol and the BK Channel

2021 ◽  
Vol 12 ◽  
Author(s):  
Sara T. Granados ◽  
Ramon Latorre ◽  
Yolima P. Torres
Keyword(s):  
Cholesterol Content ◽  
Bk Channels ◽  
Bk Channel ◽  
Cholesterol Concentration ◽  
Membrane Cholesterol ◽  
Cholesterol Depletion ◽  
Vascular Cells ◽  
Channel Activity ◽  
Channel Activation ◽  
Α Subunit

BK channels are composed by the pore forming α subunit and, in some tissues, is associated with different accessory β subunits. These proteins modify the biophysical properties of the channel, amplifying the range of BK channel activation according to the physiological context. In the vascular cells, the pore forming BKα subunit is expressed with the β1 subunit, where they play an essential role in the modulation of arterial tone and blood pressure. In eukaryotes, cholesterol is a structural lipid of the cellular membrane. Changes in the ratio of cholesterol content in the plasma membrane (PM) regulates the BK channel activation altering its open probability, and hence, vascular contraction. It has been shown that the estrogen 17β-Estradiol (E2) causes a vasodilator effect in vascular cells, inducing a leftward shift in the V0.5 of the GV curve. Here, we evaluate whether changes in the membrane cholesterol concentration modify the effect that E2 induces on the BKα/β1 channel activity. Using binding and electrophysiology assays after cholesterol depletion or enrichment, we show that the cholesterol enrichment significantly decreases the expression of the α subunit, while cholesterol depletion increased the expression of that α subunit. Additionally, we demonstrated that changes in the membrane cholesterol cause the loss of the modulatory effect of E2 on the BKα/β1 channel activity, without affecting the E2 binding to the complex. Our data suggest that changes in membrane cholesterol content could affect channel properties related to the E2 effect on BKα/β1 channel activity. Finally, the results suggest that an optimal membrane cholesterol content is essential for the activation of BK channels through the β1 subunit.

scholarly journals Cysteine Modification Alters Voltage- and Ca2+-dependent Gating of Large Conductance (BK) Potassium Channels

2005 ◽  
Vol 125 (2) ◽  
pp. 213-236 ◽  
Author(s):  
Guangping Zhang ◽  
Frank T. Horrigan
Keyword(s):  
Steady State ◽  
Bk Channels ◽  
Bk Channel ◽  
Tail Domain ◽  
Cysteine Modification ◽  
Α Subunit ◽  
Channel Opening ◽  
Rck Domain ◽  
Sensor Activation ◽  
And Shifts

The Ca2+-activated K+ (BK) channel α-subunit contains many cysteine residues within its large COOH-terminal tail domain. To probe the function of this domain, we examined effects of cysteine-modifying reagents on channel gating. Application of MTSET, MTSES, or NEM to mSlo1 or hSlo1 channels changed the voltage and Ca2+ dependence of steady-state activation. These reagents appear to modify the same cysteines but have different effects on function. MTSET increases IK and shifts the GK–V relation to more negative voltages, whereas MTSES and NEM shift the GK–V in the opposite direction. Steady-state activation was altered in the presence or absence of Ca2+ and at negative potentials where voltage sensors are not activated. Combinations of [Ca2+] and voltage were also identified where Po is not changed by cysteine modification. Interpretation of our results in terms of an allosteric model indicate that cysteine modification alters Ca2+ binding and the relative stability of closed and open conformations as well as the coupling of voltage sensor activation and Ca2+ binding and to channel opening. To identify modification-sensitive residues, we examined effects of MTS reagents on mutant channels lacking one or more cysteines. Surprisingly, the effects of MTSES on both voltage- and Ca2+-dependent gating were abolished by replacing a single cysteine (C430) with alanine. C430 lies in the RCK1 (regulator of K+ conductance) domain within a series of eight residues that is unique to BK channels. Deletion of these residues shifted the GK–V relation by >−80 mV. Thus we have identified a region that appears to strongly influence RCK domain function, but is absent from RCK domains of known structure. C430A did not eliminate effects of MTSET on apparent Ca2+ affinity. However an additional mutation, C615S, in the Haem binding site reduced the effects of MTSET, consistent with a role for this region in Ca2+ binding.

scholarly journals Molecular basis for differential modulation of BK channel voltage-dependent gating by auxiliary γ subunits

2015 ◽  
Vol 145 (6) ◽  
pp. 543-554 ◽  
Author(s):  
Qin Li ◽  
Fei Fan ◽  
Ha Rim Kwak ◽  
Jiusheng Yan
Keyword(s):  
Amino Acids ◽  
Voltage Dependence ◽  
Small Region ◽  
Bk Channels ◽  
Bk Channel ◽  
Differential Modulation ◽  
Channel Activation ◽  
Charged Amino Acids ◽  
Voltage Dependent ◽  
Positively Charged

Large conductance Ca2+- and voltage-activated potassium (BK) channels are comprised of pore-forming α subunits and various regulatory auxiliary subunits. The BK channel auxiliary γ (BKγ) subunits are a newly identified class of proteins containing an extracellular leucine-rich repeat domain (LRRD), a single transmembrane (TM) segment, and a short cytoplasmic C-terminal tail (C-tail). Although each of the four BKγ proteins shifts the voltage dependence of BK channel activation in a hyperpolarizing direction, they show markedly different efficacies, mediating shifts over a range of 15–145 mV. Analyses of chimeric BKγ subunits created by swapping individual structural elements, and of BKγ deletion and substitution mutants, revealed that differential modulation of BK gating by the four BKγ subunits depends on a small region consisting of the TM segment and the adjacent intracellular cluster of positively charged amino acids. The γ1 and γ2 TM segments contributed approximately −100 mV, and the γ1 and γ3 C-tails contributed approximately −40 mV, to shifting the voltage dependence of BK channel activation, whereas the γ3 and γ4 TM segments and the γ2 and γ4 C-tails contributed much less. The large extracellular LRRDs were mainly functionally interchangeable, although the γ1 LRRD was slightly less effective at enhancing (or slightly more effective at attenuating) the shift in BK channel voltage-dependent gating toward hyperpolarizing potentials than those of the other BKγ subunits. Analysis of mutated BKγ subunits revealed that juxta-membrane clusters of positively charged amino acids determine the functions of the γ1 and γ3 C-tails. Therefore, the modulatory functions of BKγ subunits are coarse- and fine-tuned, respectively, through variations in their TM segments and in the adjacent intracellular positively charged regions. Our results suggest that BK channel modulation by auxiliary γ subunits depends on intra- and/or juxta-membrane mechanisms.

scholarly journals Knockout of the BK β2 subunit abolishes inactivation of BK currents in mouse adrenal chromaffin cells and results in slow-wave burst activity

2014 ◽  
Vol 144 (4) ◽  
pp. 275-295 ◽  
Author(s):  
Pedro L. Martinez-Espinosa ◽  
Chengtao Yang ◽  
Vivian Gonzalez-Perez ◽  
Xiao-Ming Xia ◽  
Christopher J. Lingle
Keyword(s):  
Chromaffin Cells ◽  
Bk Channels ◽  
Bk Channel ◽  
Burst Firing ◽  
Constant Current ◽  
Repetitive Firing ◽  
Gene Encoding ◽  
Channel Activation ◽  
Α Subunit ◽  
Adrenal Chromaffin

Rat and mouse adrenal medullary chromaffin cells (CCs) express an inactivating BK current. This inactivation is thought to arise from the assembly of up to four β2 auxiliary subunits (encoded by the kcnmb2 gene) with a tetramer of pore-forming Slo1 α subunits. Although the physiological consequences of inactivation remain unclear, differences in depolarization-evoked firing among CCs have been proposed to arise from the ability of β2 subunits to shift the range of BK channel activation. To investigate the role of BK channels containing β2 subunits, we generated mice in which the gene encoding β2 was deleted (β2 knockout [KO]). Comparison of proteins from wild-type (WT) and β2 KO mice allowed unambiguous demonstration of the presence of β2 subunit in various tissues and its coassembly with the Slo1 α subunit. We compared current properties and cell firing properties of WT and β2 KO CCs in slices and found that β2 KO abolished inactivation, slowed action potential (AP) repolarization, and, during constant current injection, decreased AP firing. These results support the idea that the β2-mediated shift of the BK channel activation range affects repetitive firing and AP properties. Unexpectedly, CCs from β2 KO mice show an increased tendency toward spontaneous burst firing, suggesting that the particular properties of BK channels in the absence of β2 subunits may predispose to burst firing.

scholarly journals An S6 Mutation in BK Channels Reveals β1 Subunit Effects on Intrinsic and Voltage-dependent Gating

2006 ◽  
Vol 128 (6) ◽  
pp. 731-744 ◽  
Author(s):  
Bin Wang ◽  
Robert Brenner
Keyword(s):  
Smooth Muscle ◽  
Steady State ◽  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensor ◽  
Open Probability ◽  
Α Subunit ◽  
Channel Opening ◽  
Sensor Activation ◽  
Intracellular Domains

Large conductance, Ca2+- and voltage-activated K+ (BK) channels are exquisitely regulated to suit their diverse roles in a large variety of physiological processes. BK channels are composed of pore-forming α subunits and a family of tissue-specific accessory β subunits. The smooth muscle–specific β1 subunit has an essential role in regulating smooth muscle contraction and modulates BK channel steady-state open probability and gating kinetics. Effects of β1 on channel's gating energetics are not completely understood. One of the difficulties is that it has not yet been possible to measure the effects of β1 on channel's intrinsic closed-to-open transition (in the absence of voltage sensor activation and Ca2+ binding) due to the very low open probability in the presence of β1. In this study, we used a mutation of the α subunit (F315Y) that increases channel openings by greater than four orders of magnitude to directly compare channels' intrinsic open probabilities in the presence and absence of the β1 subunit. Effects of β1 on steady-state open probabilities of both wild-type α and the F315Y mutation were analyzed using the dual allosteric HA model. We found that mouse β1 has two major effects on channel's gating energetics. β1 reduces the intrinsic closed-to-open equilibrium that underlies the inhibition of BK channel opening seen in submicromolar Ca2+. Further, PO measurements at limiting slope allow us to infer that β1 shifts open channel voltage sensor activation to negative membrane potentials, which contributes to enhanced channel opening seen at micromolar Ca2+ concentrations. Using the F315Y α subunit with deletion mutants of β1, we also demonstrate that the small N- and C-terminal intracellular domains of β1 play important roles in altering channel's intrinsic opening and voltage sensor activation. In summary, these results demonstrate that β1 has distinct effects on BK channel intrinsic gating and voltage sensor activation that can be functionally uncoupled by mutations in the intracellular domains.

scholarly journals Hypoxic Regulation of the Large-Conductance, Calcium and Voltage-Activated Potassium Channel, BK

2021 ◽  
Vol 12 ◽  
Author(s):  
Sara V. Ochoa ◽  
Liliana Otero ◽  
Andres Felipe Aristizabal-Pachon ◽  
Fernando Hinostroza ◽  
Ingrid Carvacho ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Mrna Levels ◽  
Oxygen Balance ◽  
Channel Activation ◽  
Hypoxic Response ◽  
Α Subunit ◽  
Subunit Mrna ◽  
Obstructive Sleep

Hypoxia is a condition characterized by a reduction of cellular oxygen levels derived from alterations in oxygen balance. Hypoxic events trigger changes in cell-signaling cascades, oxidative stress, activation of pro-inflammatory molecules, and growth factors, influencing the activity of various ion channel families and leading to diverse cardiovascular diseases such as myocardial infarction, ischemic stroke, and hypertension. The large-conductance, calcium and voltage-activated potassium channel (BK) has a central role in the mechanism of oxygen (O2) sensing and its activity has been related to the hypoxic response. BK channels are ubiquitously expressed, and they are composed by the pore-forming α subunit and the regulatory subunits β (β1–β4), γ (γ1–γ4), and LINGO1. The modification of biophysical properties of BK channels by β subunits underly a myriad of physiological function of these proteins. Hypoxia induces tissue-specific modifications of BK channel α and β subunits expression. Moreover, hypoxia modifies channel activation kinetics and voltage and/or calcium dependence. The reported effects on the BK channel properties are associated with events such as the increase of reactive oxygen species (ROS) production, increases of intracellular Calcium ([Ca2+]i), the regulation by Hypoxia-inducible factor 1α (HIF-1α), and the interaction with hemeproteins. Bronchial asthma, chronic obstructive pulmonary diseases (COPD), and obstructive sleep apnea (OSA), among others, can provoke hypoxia. Untreated OSA patients showed a decrease in BK-β1 subunit mRNA levels and high arterial tension. Treatment with continuous positive airway pressure (CPAP) upregulated β1 subunit mRNA level, decreased arterial pressures, and improved endothelial function coupled with a reduction in morbidity and mortality associated with OSA. These reports suggest that the BK channel has a role in the response involved in hypoxia-associated hypertension derived from OSA. Thus, this review aims to describe the mechanisms involved in the BK channel activation after a hypoxic stimulus and their relationship with disorders like OSA. A deep understanding of the molecular mechanism involved in hypoxic response may help in the therapeutic approaches to treat the pathological processes associated with diseases involving cellular hypoxia.

scholarly journals Deletion of cytosolic gating ring decreases gate and voltage sensor coupling in BK channels

2017 ◽  
Vol 149 (3) ◽  
pp. 373-387 ◽  
Author(s):  
Guohui Zhang ◽  
Yanyan Geng ◽  
Yakang Jin ◽  
Jingyi Shi ◽  
Kelli McFarland ◽  
...  
Keyword(s):  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensor ◽  
Voltage Sensitivity ◽  
Gating Currents ◽  
Voltage Sensors ◽  
Transmembrane Voltage ◽  
Wide Range ◽  
Activation Gate ◽  
Sensor Activation

Large conductance Ca2+-activated K+ channels (BK channels) gate open in response to both membrane voltage and intracellular Ca2+. The channel is formed by a central pore-gate domain (PGD), which spans the membrane, plus transmembrane voltage sensors and a cytoplasmic gating ring that acts as a Ca2+ sensor. How these voltage and Ca2+ sensors influence the common activation gate, and interact with each other, is unclear. A previous study showed that a BK channel core lacking the entire cytoplasmic gating ring (Core-MT) was devoid of Ca2+ activation but retained voltage sensitivity (Budelli et al. 2013. Proc. Natl. Acad. Sci. USA. http://dx.doi.org/10.1073/pnas.1313433110). In this study, we measure voltage sensor activation and pore opening in this Core-MT channel over a wide range of voltages. We record gating currents and find that voltage sensor activation in this truncated channel is similar to WT but that the coupling between voltage sensor activation and gating of the pore is reduced. These results suggest that the gating ring, in addition to being the Ca2+ sensor, enhances the effective coupling between voltage sensors and the PGD. We also find that removal of the gating ring alters modulation of the channels by the BK channel’s β1 and β2 subunits.

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